Restriction Endonuclease Digestion of Plasmid Dna

Introduction: With the execution of this experiment, we began to go deeper into the Cell and Molecular Biology course. The main focus of the experiment would be how the Restriction Endonucleases cleave the strands of DNA. For this experiment, pBR322 was the specimen to use. Restriction Endonucleases work by cleaving the sugar phosphate backbone of specific DNA sites. Restriction enzymes that have been isolated from bacteria have a defensive role. This idea is illustrated when an attacking foreign cell DNA is trying to alter the bacteria; restriction enzymes cleave the DNA rendering it inert.

The second part of the experiment deals with Gel Electrophoresis. The samples are loaded into wells on an 1% agarose slab and subjected to electrical currents both positive and negative. Our current target here is DNA, therefore since nucleic acid as a negative charge, the bands will migrate toward the positive cathode. This process of migration is called sieving and smaller strands move faster than longer strands due to their ease in going through the gel.

The objectives of the experiment include:Learning the principles behind Restriction Enzymes and Gel Electrophoresis Applying the concepts in the experiment to produce bands at the end of the Gel Electrophoresis stage Interpreting what these bands mean with accordance to how the plasmid was cleaved

The first procedure began by adding 8.5 µL sterile distilled H2O, 1.0µL of the appropriate 10x buffer, 1.0µL combination of the restriction endonucleases and 1.0µL of pBR322 plasmid DNA (the DNA would be added last) in 5...

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...stranded DNA ¡VdsDNA). The DNA of the phage contains around 50,000 base pairs and codes for 50 proteins. At both ends of the DNA of the phage, there are cohesive ends, which are composed of 12 nucleotides. Both ends compliment each other, which makes the DNA circular once together. This circular DNA is usually present in an infected bacterial cell. This protects the DNA to be degraded by the cell.
In this laboratory experiment, restriction enzymes were used to analyze the DNA of the lambda phage. Restriction enzymes cut DNA in specific locations of the DNA sequence. The restriction enzymes used were EcoR1 and Bam H1. Both of these restriction enzymes cut DNA into six fragments.
Materials
Agarose
Gel Electrophoresis Apparatus
Gel Comb
Gel Mold
100x Triacetate EDTA Buffer with a pH of 8.0
Methylene Blue Stain
Microwave
Micropipette
Ice Bath
Water Bath 37¢XC (Incubator)
Water Bath 70¢XC (Incubator)
Phage Lambda DNA (uncut)
EcoR1- Restriction Enzyme
EcoR1 + Bam H1 ¡V Mixture of Restriction Enzymes
Distilled Water
Methods
¡÷ Preparing the DNA samples
In this lab experiment, four small tubes were labeled 1-4. Once labeled, 10£gl of the following material was placed in each of the tubes as shown...

...
Objective:
DNA is analyzed by agarose gel electrophoresis after being digested with EcoRI restriction endonucleasse.
Procedures:
λ DNA and puC18 DNA were put into two tubes respectively. Then, EcoRI buffer, EcoRI enzyme and deionized water would be put into both tubes. EcoRI enzyme was the restriction enzyme that cut the DNA at the specific sequence. The EcoRI buffer enhanced the stability of many enzymes and binds contaminants that may be present in DNA preparations. DI water was used to bring the solution into a required volume for gel electrophoresis. The prepared sample was incubated at 37℃ for overnight.
40mL of a 0.8% agarose gel is prepared after the preparation of the samples was done. Agarose and 1X TAE electrophoresis buffer were added into an Erlenmeyer flask. The 1X TAE electrophoresis buffer provided the correct pH for the gel to run. The solution would be heat until the agarose is completely dissolved. Ethidium bromide solution would be added to the solution after it cooled slightly. Ethidium bromide solution was a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments. It enabled visualization of the fragments within the gel under UV light. The solution would be poured into the gel mold for solidification. The gel was stored at 4℃ until used.
All the samples and the gel...

...DNADIGESTION AND ELECTROPHORESIS
In this experiment we will be doing a process called as DNAdigestion or also known as restriction digest. A restriction digest is a procedure used in molecular biology to prepare DNA for analysis or other processing. It is sometimes termed DNA fragmentation, scientists Hartl and Jones describe it this way:
This enzymatic technique can be used for cleaving DNA molecules at specific sites, ensuring that all DNA fragments that contain a particular sequence have the same size; furthermore, each fragment that contains the desired sequence has the sequence located at exactly the same position within the fragment. The cleavage method makes use of an important class of DNA-cleaving enzymes isolated primarily from bacteria. These enzymes are called restrictionendonucleases or restriction enzymes, and they are able to cleave DNA molecules at the positions at which particular short sequences of bases are present.
The resulting digested DNA is very often selectively amplified using PCR, making it more suitable for analytical techniques such as agarose gel electrophoresis, andchromatography. It is used in genetic fingerprinting, and RFLP analysis. [1]
Just as mentioned above, for this experiment we will be using...

...Isolation of plasmid-DNA from bacteria and PCR
Advisor
Konrad Egli: kegli@botinst.unizh.ch
Reading
Chapters in BBOM 10th: 10.8
BBOM : Madigan M.T., J.M. Martinko and J. Parker: "Brock - Biology of
Microorganisms", 10th Edition (2003), Prentice Hall.
Objectives
Background
• Isolation of plasmid-DNA from different bacteria clones
• Handling of bacteria clones
• PCR-experiment
The typical plasmid is a circular double-standed DNA molecule less than 1/20 the
size of the chromosome. Most plasmids are circular, but linear plasmids are also
known. Naturally occuring plasmids vary in size from 1 to more than 1000 kilobase
pairs. Most of the plasmid-DNA isolated from cells is in the supercoiled
configuration, which is the most compacted form within the cell.
Isolation of plasmid-DNA can generally be accomplished by making use of the
physical properties of supercoiled DNA molecules. Although chromosomes are also
supercoiled inside the cell, isolation of chromosomal DNA almost always leads to
breakage of the strands and consequent loss of supercoiling. Separation can then
proceed by a variety of techniques, including ultracentrifugation and electrophoresis
on agarose gels.
Among the most widespread and well-studied groups of...

...Isolation of plasmidDNA and analysis of isolated plasmid
Introduction: A plasmid is an autonomously replicating extra-chromosomal genetic element. In other words, this is a DNA molecule external to the bacterial chromosome that is able to replicate on its own and distribute its daughter molecules to daughter cells. You have successfully cloned a fragment of chromosomal DNA containing a tetracycline resistance cassette into a plasmid (pET11a). To this end you have (1) isolated total chromosomal DNA from your assigned bacterial strain and digested this DNA with the restriction enzyme BamHI. You have also (2) digested the plasmid cloning vector (pET11a) with the same restriction enzyme. You have (3) randomly ligated the digested chromosomal DNA into the plasmid and (4) transformed an Escherichia coli strain JM109. You have selected for transformants by plating the transformed DNA onto rich media (TCS) containing ampicillin. (The pET11a plasmid contains an ampicillin resistance cassette that renders any bacteria harboring this plasmid resistant to ampicillin and therefore capable of growing on media containing ampicillin). Finally, you have (5) scored for transformants that are not only resistant to ampicillin but also resistant...

...DNADNA, or Deoxyribonucleic Acid, is described, in Encarta Encyclopedia as a genetic material of all cellular organisms and most viruses. DNA carries the information needed to direct protein synthesis and replication. Protein synthesis is the production of the proteins needed by the cell or virus for its activities and development. Replication is the process by which DNA copies itself for each descendant cell or virus, passing on the information needed for protein synthesis. In most cellular organisms, DNA is organized on chromosomes located in the nucleus of the cell.
A molecule of DNA consists of two chains, strands composed of a large number of nucleotides, that are linked together to form a chain. These chains look like a twisted ladder and are called a double helix. Each nucleotide consists of three units: sugar molecules called deoxyribose, a phosphate group, and one of four different nitrogen containing compounds, also called bases. The four are adenine (A), guanine (G), thymine (T), and cytosine (C). The deoxyribose molecule occupies the center of the nucleotide, with the phosphate group on one side and a base on the other. The phosphate group of each nucleotide is also linked to the deoxyribose of the adjacent nucleotide in the chain. These linked deoxyribose-phosphate subunits form the side rails of the ladder. The bases face inward toward each other, forming the steps of...

...Introduction
Plasmids are circular, double stranded extrachromosomal DNA molecules that are found in bacteria which can self-replicate. They are naturally occurring DNA molecules advantageous to the host bacterium by carrying genes which specify metabolic capacities. (Garrett et al., 2010) Besides, plasmids exist in a wide variety of sizes from a few thousands to hundreds of thousands of base pairs. Many plasmids have been engineered to serve as plasmids cloning vectors to carry genes. (Synder et al., 2007) Useful plasmids that serve as cloning vectors have three common features which are an origin of replication, a selectable marker and a cloning site. (Garrett et al., 2010) Plasmid pBR322 is an example of one of the widely used cloning vector that has 4363 base pairs. This plasmid has an origin of replication, a selectable marker which codes for ampicillin and tetracycline resistance gene, and a multiple cloning site.
The structure of plasmids is mostly circular with no ends, except with a few known plasmids which are linear. Plasmids can also be supercoiled in structure when the strands are prevented from separating and there are no ends to rotate (Synder et al., 2007). Three conformations that plasmidDNA can exist in are supercoiled, open-circular (nicked), and linear....

...DIFFERENCES BETWEEN PLASMID AND CHROMOSOMAL DNA IN BACTERIA.
Eukaryotes have two or more chromosomes, prokaryotes such as bacteria possess a single chromosome composed of double-stranded DNA in a loop. DNA is located in the nucleoid of the cell and is not associated with protein
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. Plasmids usually occur naturally in bacteria.
A chromosome is an organized structure of DNA and protein that is found in cells. A chromosome is a single piece of coiled DNA containing many genes, regulatory elements and other nucleotide sequences. Chromosomes also contain DNA-bound proteins, which serve to package the DNA and control its functions.
1. Plasmids are circular and double stranded.
2. Plasmids have much less base pairs than chromosomes
3. Plasmids are rarely organized by chaperone proteins
4. Plasmids are easily transferred
- Plasmids are considered transferable genetic elements, or "replicons", capable of
autonomous replication within a suitable host. Plasmids can be removed from the host
cell in...